Stroke is a major cause of morbidity and mortality in the United States. To help understand how brain cells die during stroke, it is important to understand the effect of hypoxia/ischemia on neuronal function, for example, how it affects synaptic activity. BCL-2 family proteins are known to regulate cell death during development of the nervous system, and have recently been shown to influence the fate of neurons after insults to me nervous system such as hypoxia/ischemia. BCL-2 family proteins are present in mitochondrial membranes and it is there, in the oxygen-sensing organelle of the neuron that these proteins may respond to a low concentration of cellular oxygen. The response by BCL-2 family proteins includes the activation of ion channel activity in mitochondrial membranes, but the exact mechanism and consequences of this activation are not yet understood. Using a technique that we developed, we will study the ion channel activity of BCL-2 family proteins in intact, living presynaptic terminals, in order to attempt to understand how opening of ion channels produced by actions of a pro-apoptotic version of BCL-XL can lead to loss of release of neurotransmitter during hypoxia. Recent findings of our laboratory have shown that, during a hypoxic insult to a synapse, rundown of neurotransmission is related to the opening of a large channel on the outer membranes of presynaptic mitochondria. Additional studies from our laboratory have shown that BCL-xL injection into a presynaptic terminal causes potentiation of neurotransmission and that this potentiation can be mimicked by or occluded by injection into the synapse of ATP. We therefore hypothesize that, apart from their role in cell death, BCL-2 family proteins serve a unique function in the synapse by regulating neurotransmitter release through mitochondrial ATP availability. Specific aims are to 1) Determine if cleavage of BCL-xL contributes to the formation of large conductance mitochondrial membrane channels early in the response of neurons to hypoxia. 2) Determine if the mechanism of formation of large conductance channels requires the voltage dependent anion channel (VDAC) of the outer mitochondnal channel. 3) Determine the mechanism of synaptic rundown in hypoxia by testing the hypothesis that either a cleaved pro-apoptotic form of BCL-xL or hypoxia decreases the availability of ATP that is required for synaptic transmission. 4) Determine the mechanism of potentiation or synaptic transmission by full length BCL-xL by testing the hypothesis that BCLxL causes an increase in concentration of free cytoplasmic ATP in the synapse. The overall goal of the studies will be to understand how tight regulation of mitochondrial ion channels in the synapse leads to loss of, or potentiation of, synaptic transmission, and how regulation of synaptic strength may be tied to regulation of the life and death of a neuron. The studies may also lead to new insights in the field of learning and memory.